PROJECT SUMMARY The prevalence of chronic kidney disease (CKD), which may progress to end-stage renal disease (ESRD) and kidney failure, continues to rise worldwide. This increase in prevalence is due in part to the heightened incidence of contributing medical conditions such as diabetes and hypertension that lead to progressive damage to the capillaries and basement membrane of the kidney glomerulus. As a result, there is a compelling need for bioengineered kidney tissues as models for organ development, disease, or drug discovery and as future substitutes as reparative or replacement therapies. The glomerulus, as the first segment of the nephron, is where blood initially interfaces with and is filtered by the kidney. In the glomerulus, glomerular endothelial cells (GEnCs) and podocytes closely interact to form and maintain the glomerular filtration barrier. One critical signaling factor mediating this interaction is vascular endothelial growth factor (VEGF), which is secreted by podocytes. This VEGF expression is necessary to recruit endothelial progenitors during nephrogenesis and is additionally maintained in the mature kidney. Currently, the complex cell-cell and cell- matrix interactions within the glomerulus remain poorly understood, limiting our ability to recreate the glomerular filtration barrier and engineer or reconstruct the glomerulus. For that reason, the goal of this project is to investigate microenvironmental signaling cues hypothesized to be necessary for recapitulating cell-cell and cell-matrix interactions within the native glomerulus. VEGF secreted by podocytes may be sequestered within the glomerular basement membrane, the layer of extracellular matrix (ECM) deposited by both GEnCs and podocytes. Therefore, Specific Aim 1 will investigate the hypothesis that ECM-bound VEGF is critical to enhance GEnC and podocyte response and reciprocal interaction. ECM will be isolated from the kidneys of transgenic mice in which VEGF secretion by podocytes is abrogated or abolished. GEnCs and podocytes will be cultured on the isolated kidney ECM, and cellular response to measured levels of matrix-bound VEGF will be evaluated. Specific Aim 2 will investigate the hypothesis that bioactive composite ECM hydrogel scaffolds with controlled spatial architecture will upregulate development of glomerular endothelial tube networks and podocyte foot processes. The spatial architecture of the native glomerulus is unique, and as a result controlled spatial architecture of hydrogels may provide additional signaling cues to guide GEnC and podocyte morphogenesis and maturation. Composite biomaterial hydrogels of gelatin and isolated kidney ECM material will be formulated and tuned for 3D printability. From printable formulations, hydrogel scaffolds will be fabricated via 3D printing to precisely control the spatial architecture of the matrix environment. GEnCs and podocytes will be cultured on scaffolds, and cellular response will be compared to culture on cast hydrogels. Ultimately, the results from these investigations will enhance our understanding of cell-cell and cell-matrix interactions within the glomerulus and advance engineering of complex tissues and organs such as the kidney for scientific and clinical applications.